DESCRIPTION (APPLICANT'S ABSTRACT): Chromosome segregation is a critical process necessary for normal development and genetic stability. Defects in this process lead to aneuploidy, a direct cause of the developmental disorder Down syndrome and a contributing factor in most cancers. The centromere is a unique region on each chromosome that specifies chromosome attachment to the mitotic spindle, chromosome motility in mitosis and meiosis and critical signal transduction pathways that monitor and maintain normal chromosome number. Recent findings have revealed that the chromatin assembly pathway used to replicate centromeres is distinct from that which operates on the chromosome arms. The work of this proposal will test the hypothesis that centromere identity involves a critical seif-templated chromatin assembly pathway by identifying chromatin assembly factors that assemble a centromere-specific chromatin domain that substitutes the core histone H3 for a centromere specific H3-like protein known as CENP-A. Chromatin complexes assembled in vitro will be introduced into human HT1080 cells to directly determine whether CENP-A and/or other centromere-specific chromatin factors template their own deposition during DNA replication and chromatin assembly in vivo. The molecular structure and composition of centromere-specific chromatin comprised of CENP-A nucleosomes will be dissected by direct purification of CENP-A chromatin complexes followed by mass spectrometric identification of associated proteins, by X-ray crystallography of reconstituted CENP-A nucleosomes and by electron microscopy of reconstituted CENP-A chromatin fibers. Post-translational modifications of CENP-A will be characterized to determine whether centromeric chromatin is subject to similar kinds of post-translational control as observed for non-centromeric chromatin. The function of CENP-A will be examined by developing knock out and knock down models in mouse and cultured human cells coupled with mutational analysis of CENP-A to explore the specific role of this histone homologue in centromere assembly and function.